Neuroprotective effects of theobromine in transient global cerebral ischemia-reperfusion rat model.

Transient global cerebral ischemia (tGCI) is a cerebrovascular disorder characterized by a brief decline in blood flow, neurological deficits, and is often predictive of stroke. Theobromine (TBR) is consumed worldwide in chocolates, tea, and cocoa products. TBR is a natural stimulant and vasoactive alkaloid that may protect against ischemic injury. In this study, neuroprotective potential of theobromine (TBR) was evaluated in 2-vessel occlusion transient global cerebral ischemia-reperfusion (tGCI/R) rat model. Rats were treated with TBR (50, 100 mg/kg, p.o.) for 7 successive days, and subjected to bilateral common carotid artery occlusion (20 min) or sham surgery after last dose of TBR. Severe neurological deficits accompanied by brain infarction, blood-brain barrier abnormalities, and oedema were noted in rats subjected to tGCI/R, and these effects were prevented by TBR. TBR protected against lipid peroxidation and enhanced glutathione level in brain against tGCI/R. TBR pre-treatment for 7 days prevented tGCI/R induced cell death (lactate dehydrogenase, caspase-3), vascular injury (MMP-9), and inflammation (TNF-α, NFκB) in rat whole brain. TBR protected against glutamate excitotoxicity and GABAergic decline in the brain of rats against tGCI/R injury. Findings of this study showed that TBR can alleviate chances of stroke by preventing acute episodes of cerebral ischemia.

Methylxanthines and Neurodegenerative Diseases: An Update.

Methylxanthines (MTX) are purine derived xanthine derivatives. Whereas naturally occurring methylxanthines like caffeine, theophylline or theobromine are widely consumed in food, several synthetic but also non-synthetic methylxanthines are used as pharmaceuticals, in particular in treating airway constrictions. Besides the well-established bronchoprotective effects, methylxanthines are also known to have anti-inflammatory and anti-oxidative properties, mediate changes in lipid homeostasis and have neuroprotective effects. Known molecular mechanisms include adenosine receptor antagonism, phosphodiesterase inhibition, effects on the cholinergic system, wnt signaling, histone deacetylase activation and gene regulation. By affecting several pathways associated with neurodegenerative diseases via different pleiotropic mechanisms and due to its moderate side effects, intake of methylxanthines have been suggested to be an interesting approach in dealing with neurodegeneration. Especially in the past years, the impact of methylxanthines in neurodegenerative diseases has been extensively studied and several new aspects have been elucidated. In this review we summarize the findings of methylxanthines linked to Alzheimer´s disease, Parkinson's disease and Multiple Sclerosis since 2017, focusing on epidemiological and clinical studies and addressing the underlying molecular mechanisms in cell culture experiments and animal studies in order to assess the neuroprotective potential of methylxanthines in these diseases.

Lipotoxicity-associated inflammation is prevented by guarana (Paullinia cupana) in a model of hyperlipidemia.

Hyperlipidemia causes lipotoxicity which prompts an inflammatory response linked to the development of cardiovascular diseases. Natural compounds have been receiving special attention for its potential to treat diseases, inexpensiveness, and safety. Guarana (Paullinia cupana) has demonstrated notable anti-inflammatory and antioxidant effects, which may prevent chronic diseases caused by changes in lipid profile. Thus, this study aims to evaluate the effect of guarana powder (Paullinia cupana) in the purine metabolism and inflammatory profile in lymphocytes and serum of rats with Poloxamer-407-induced hyperlipidemia. Pretreatment with guarana 12.5, 25, and 50 mg/kg/day or caffeine (0.2 mg/kg/day) by gavage was applied to adult male Wistar rats for a period of 30 days. As a comparative standard, we used simvastatin (0.04 mg/kg) post-induction. Hyperlipidemia was acutely induced with intraperitoneally injection of Poloxamer-407 (500 mg/kg). Guarana powder and caffeine increased the activity of the E-NTPDase (ecto-apyrase), and all pretreatments decreased the E-ADA (ecto-adenosine deaminase) activity, reducing the inflammatory process caused by lipotoxicity. In hyperlipidemic rats, ATP levels were increased while adenosine levels were decreased, guarana and caffeine reverted these changes. Guarana powder, caffeine, and simvastatin also prevented the increase in INF-γ and potentiated the increase in IL-4 levels, promoting an anti-inflammatory profile. Guarana promoted a more robust effect than caffeine. Our results show that guarana powder and caffeine have an anti-inflammatory as seen by the shift from a proinflammatory to an anti-inflammatory profile. The effects of guarana were more pronounced, suggesting that guarana powder may be used as a complementary therapy to improve the lipotoxicity-associated inflammation.

Chronic administration of theobromine inhibits mTOR signal in rats.

Theobromine is a caffeine derivative and the primary methylxanthine in Theobroma cacao. We have shown previously that theobromine inhibits the Akt-mammalian target of rapamycin (mTOR) signal in vitro. In this study, we investigated whether orally administered theobromine could inhibit mTOR activity in rats. mTOR is phosphorylated by Akt. Thus, the level of phosphorylated mTOR was used as an index of mTOR activity. Male Wistar rats were divided into two groups. The control group (CN) was fed a normal diet, while the theobromine group (TB) was fed a diet supplemented with 0.05% theobromine for 40 days. We measured body-weights and tissue weights, food and water intake, blood count, concentrations of theobromine in the plasma, liver and brain, and the levels of phosphorylated mTOR in the liver and brain. Orally administered theobromine did not affect the body-weights and tissue weights, food and water intake, and blood count as determined by comparison with levels in rats that were fed standard chow. Theobromine was detected in the plasma, liver and brain obtained from TB rats, but was not detected in tissues obtained from CN rats. The phosphorylated mTOR levels in the liver and brain were significantly lower in TB rats than in CN rats. The results suggest that oral theobromine inhibits mTOR signalling in vivo.

Antidepressant-Like Activity of Typical Antidepressant Drugs in the Forced Swim Test and Tail Suspension Test in Mice Is Augmented by DMPX, an Adenosine A2A Receptor Antagonist.

Unsatisfactory therapeutic effects of currently used antidepressants force to search for new pharmacological treatment strategies. Recent research points to the relationship between depressive disorders and the adenosinergic system. Therefore, the main goal of our studies was to evaluate the effects of DMPX (3 mg/kg, i.p.), which possesses selectivity for adenosine A2A receptors versus A1 receptors, on the activity of imipramine (15 mg/kg, i.p.), escitalopram (2.5 mg/kg, i.p.), and reboxetine (2 mg/kg, i.p.) given in subtherapeutic doses. The studies carried out using the forced swim and tail suspension tests in mice showed that DMPX at a dose of 6 and 12 mg/kg exerts antidepressant-like effect and does not affect the locomotor activity. Co-administration of DMPX at a dose of 3 mg/kg with the studied antidepressant drugs caused the reduction of immobility time in both behavioral tests. The observed effect was not associated with an increase in the locomotor activity. To evaluate whether the observed effects were due to a pharmacokinetic/pharmacodynamic interaction, the levels of the antidepressants in blood and brain were measured using high-performance liquid chromatography. It can be assumed that the interaction between DMPX and imipramine was exclusively pharmacodynamic in nature, whereas an increased antidepressant activity of escitalopram and reboxetine was at least partly related to its pharmacokinetic interaction with DMPX.

The acute effects on duodenal gene expression in healthy men following consumption of a low-fat meal enriched with theobromine or fat.

Increasing apoA-I synthesis may improve HDL functionality and lower CVD risk. As theobromine and fat increase fasting apoA-I concentrations, and the intestine is involved in apoA-I production, the acute effects of both were studied on duodenal gene transcription to better understand underlying mechanisms. In this crossover study, 8 healthy men received once a low fat (LF) meal, a LF meal plus theobromine (850 mg), or a high fat (HF) meal. Five hours after meal intake duodenal biopsies were taken for microarray analysis. Theobromine and HF consumption did not change duodenal apoA-I expression. Theobromine did not change gene expression related to lipid and cholesterol metabolism, whereas those related to glycogen/glucose breakdown were downregulated. HF consumption increased gene expression related to lipid and cholesterol uptake and transport, and to glucose storage, while it decreased those related to glucose uptake. Furthermore, genes related to inflammation were upregulated, but inflammation markers in plasma were not changed. In healthy men, acute theobromine and fat consumption did not change duodenal apoA-I mRNA, but inhibited expression of genes related to glucose metabolism. Furthermore, HF intake activated in the duodenum expression of genes related to lipid and cholesterol metabolism and to inflammation.

Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein ß degradation by adenosine receptor A1.

Theobromine, a methylxanthine derived from cacao beans, reportedly has various health-promoting properties but molecular mechanism by which effects of theobromine on adipocyte differentiation and adipogenesis remains unclear. In this study, we aimed to clarify the molecular mechanisms of the anti-adipogenic effect of theobromine in vitro and in vivo. ICR mice (4week-old) were administered with theobromine (0.1g/kg) for 7days. Theobromine administration attenuated gains in body and epididymal adipose tissue weights in mice and suppressed expression of adipogenic-associated genes in mouse adipose tissue. In 3T3-L1 preadipocytes, theobromine caused degradation of C/EBPß protein by the ubiquitin-proteasome pathway. Pull down assay showed that theobromine selectively interacts with adenosine receptor A1 (AR1), and AR1 knockdown inhibited theobromine-induced C/EBPß degradation. Theobromine increased sumoylation of C/EBPß at Lys133. Expression of the small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2) gene, coding for a desumoylation enzyme, was suppressed by theobromine. In vivo knockdown studies showed that AR1 knockdown in mice attenuated the anti-adipogenic effects of theobromine in younger mice. Theobromine suppresses adipocyte differentiation and induced C/EBPß degradation by increasing its sumoylation. Furthermore, the inhibition of AR1 signaling is important for theobromine-induced C/EBPß degradation.

The effect of a single dose of multivitamin and mineral combinations with and without guaraná on functional brain activity during a continuous performance task.

OBJECTIVES: Relatively few studies have explored the possibility of acute cognitive effects of multivitamin ingestion. This report explores the acute brain electrophysiological changes associated with multivitamin and mineral supplementation, with and without guaraná, using the steady-state visually evoked potential (SSVEP). METHODS: Based on the known SSVEP correlates of A-X continuous performance task (CPT) performance, and sensitivity to acute psychopharmacological manipulations, the A-X CPT was adopted as a task paradigm to explore treatment-related neurophysiological changes in attentional processing. Twenty healthy non-smoking adults aged 21-39 years (mean age = 28.35 years, SD = 5.52) took part in this double-blind, placebo-controlled, randomized, balanced crossover design study. RESULTS: The study demonstrated both transient and tonic changes in the SSVEP response during completion of the A-X CPT following multivitamin and mineral treatment both with and without guaraná. Transient changes in SSVEP response in prefrontal regions were observed after a single dose of a multivitamin and mineral preparation indicative of enhanced activity within brain regions engaged by the attentional demands of the task. This pattern of change in frontal regions was correlated with improved behavioural performance after treatment with the multivitamin and mineral combination. Where tonic shifts in SSVEP response were investigated, multivitamin and mineral treatment was associated with a pattern of increased inhibition across posterior regions, with enhanced excitatory processing in prefrontal regions. In contrast, multivitamin and mineral treatment with additional guaraná showed a tonic shift towards greater excitatory processes after a single treatment, consistent with the caffeine content of this treatment. DISCUSSION: While preliminary in nature, these findings suggest a single multivitamin/mineral dose is sufficient to impact on functional brain activity in task-related brain regions.

Theobromine-Induced Changes in A1 Purinergic Receptor Gene Expression and Distribution in a Rat Brain Alzheimer's Disease Model.

Dementia caused by Alzheimer's disease (AD) is mainly characterized by accumulation in the brain of extra- and intraneuronal amyloid-ß (Aß) and tau proteins, respectively, which selectively affect specific regions, particularly the neocortex and the hippocampus. Sporadic AD is mainly caused by an increase in apolipoprotein E, a component of chylomicrons, which are cholesterol transporters in the brain. Recent studies have shown that high lipid levels, especially cholesterol, are linked to AD. Adenosine is an atypical neurotransmitter that regulates a wide range of physiological functions by activating four P1 receptors (A1, A2A, A2B, and A3) and P2 purinergic receptors that are G protein-coupled. A1 receptors are involved in the inhibition of neurotransmitter release, which could be related to AD. The aim of the present work was to study the effects of a lard-enriched diet (LED) on cognitive and memory processes in adult rats (6 months of age) as well as the effect of theobromine on these processes. The results indicated that the fat-enriched diet resulted in a long-term deterioration in cognitive and memory functions. Increased levels of Aß protein and IL-1ß were also observed in the rats fed with a high-cholesterol diet, which were used to validate the AD animal model. In addition, the results of qPCR and immunohistochemistry indicated a decrease in gene expression and distribution of A1 purinegic receptor, respectively, in the hippocampus of LED-fed rats. Interestingly, theobromine, at both concentrations tested, restored A1 receptor levels and improved cognitive functions and Aß levels for a dose of 30 mg/L drinking water.

Methylxanthines enhance the effects of cocoa flavanols on cardiovascular function: randomized, double-masked controlled studies.

BACKGROUND: Cocoa flavanol intake, especially that of (-)-epicatechin, has been linked to beneficial effects on human cardiovascular function. However, cocoa also contains the methylxanthines theobromine and caffeine, which may also affect vascular function. OBJECTIVE: We sought to determine whether an interaction between cocoa flavanols and methylxanthines exists that influences cocoa flavanol-dependent vascular effects. DESIGN: Test drinks that contained various amounts of cocoa flavanols (0-820 mg) and methylxanthines (0-220 mg), either together or individually, were consumed by healthy volunteers (n = 47) in 4 different clinical studies-3 with a randomized, double-masked crossover design and 1 with 4 parallel crossover studies. Vascular status was assessed by measuring flow-mediated vasodilation (FMD), brachial pulse wave velocity (bPWV), circulating angiogenic cells (CACs), and blood pressure before and 2 h after the ingestion of test drinks. RESULTS: Although cocoa flavanol intake increased FMD 2 h after intake, the consumption of cocoa flavanols with methylxanthines resulted in a greater enhancement of FMD. Methylxanthine intake alone did not result in statistically significant changes in FMD. Cocoa flavanol ingestion alone decreased bPWV and diastolic blood pressure and increased CACs. Each of these changes was more pronounced when cocoa flavanols and methylxanthines were ingested together. It is important to note that the area under the curve of the plasma concentration of (-)-epicatechin metabolites over time was higher after the co-ingestion of cocoa flavanols and methylxanthines than after the intake of cocoa flavanols alone. Similar results were obtained when pure (-)-epicatechin and the methylxanthines theobromine and caffeine were consumed together. CONCLUSION: A substantial interaction between cocoa flavanols and methylxanthines exists at the level of absorption, in which the methylxanthines mediate an increased plasma concentration of (-)-epicatechin metabolites that coincides with enhanced vascular effects commonly ascribed to cocoa flavanol intake. This trial was registered at clinicaltrials.gov as NCT02149238.

Catecholaminergic and cholinergic systems of mouse brain are modulated by LMN diet, rich in theobromine, polyphenols and polyunsaturated fatty acids.

The possible modulatory effect of the functional LMN diet, rich in theobromine, polyphenols and polyunsaturated fatty acids, on the catecholaminergic and cholinergic neurotransmission, affecting cognition decline during aging has been studied. 129S1/SvlmJ mice were fed for 10, 20, 30 and 40 days with either LMN or control diets. The enzymes involved in catecholaminergic and cholinergic metabolism were determined by both immunohistological and western blot analyses. Noradrenalin, dopamine and other metabolites were quantified by HPLC analysis. Theobromine, present in cocoa, the main LMN diet component, was analysed in parallel using SH-SY5Y and PC12 cell lines. An enhanced modulatory effect on both cholinergic and catecholaminergic transmissions was observed on 20 day fed mice. Similar effect was observed with theobromine, besides its antioxidant capacity inducing SOD-1 and GPx expression. The enhancing effect of the LMN diet and theobromine on the levels of acetylcholine-related enzymes, dopamine and specially noradrenalin confirms the beneficial role of this diet on the "cognitive reserve" and hence a possible reducing effect on cognitive decline underlying aging and Alzheimer's disease.

Effects of theobromine and caffeine on mood and vigilance.

Like caffeine, theobromine crosses the blood-brain barrier and binds to adenosine receptors, suggesting it might share caffeine's beneficial effects on mood and vigilance. Therefore, the purpose of this study was to assess the effect of theobromine doses commonly found in foods on mood and vigilance parameters sensitive to caffeine. Caffeine was tested as a positive control. Twenty-four men (age, 23 [3] years) completed 6 double-blind trials during which they consumed experimental beverages, assessed their mood using standardized self-report questionnaires, and completed a 2-hour visual vigilance task. Three experimental doses (100, 200, and 400 mg theobromine) were delivered in a cocoa-based beverage; 3 matched control treatments (0 mg theobromine, 400 mg theobromine, and 100 mg caffeine) were delivered in a non-cocoa beverage. Mean salivary concentrations of theobromine exhibited significant dose-dependent differences (400 mg trials > 200 mg trial > 100 mg trial > 0 mg trials; P < 0.005). At every dose tested, theobromine failed to consistently affect mood state or vigilance (P > 0.05), but 100-mg caffeine significantly decreased lethargy/fatigue and increased vigor (P = 0.006 and 0.011, respectively). These findings indicate theobromine does not influence mood and vigilance when administered in nutritionally relevant doses, despite sharing many of caffeine's structural characteristics.

Hypotensive effects of intravenously administered uridine and cytidine in conscious rats: involvement of adenosine receptors.

In the present study, we investigated the cardiovascular effects of intravenously injected uridine or cytidine, and the role of adenosine receptors in mediating these effects, in conscious normotensive rats. Intravenous (i.v.) administration of uridine (124, 250, 500 mg/kg) dose-dependently decreased arterial pressure and heart rate. Cytidine (124, 250, 500 mg/kg; i.v.) produced slight dose-related hypotension without changing heart rate. Plasma uridine and cytidine concentrations increased time- and dose-dependently while plasma adenosine levels did not change after injection of the respective nucleosides. Pretreatment with intravenous caffeine (20 mg/kg), 8-phenyltheophylline (8-PT) (1 mg/kg), nonselective adenosine receptor antagonists, or 8-p-sulfophenyltheophylline (8-SPT) (20 mg/kg), a nonselective adenosine receptor antagonist which does not cross the blood-brain barrier, abolished the cardiovascular effects of uridine (250 mg/kg; i.v.) or cytidine (250 mg/kg; i.v.). Intracerebroventricular (i.c.v.) caffeine (200 microg) or 8-SPT (50 microg) pretreatment did not change the magnitude of the cardiovascular responses induced by nucleosides. Intravenous 8-cyclopenthyl-1,3-dipropylxanthine (DPCPX) (5 mg/kg), a selective adenosine A(1) receptor antagonist, greatly attenuated the cardiovascular responses to uridine and cytidine. Pretreatment with 3,7,-dimethyl-1-propargylxanthine (DMPX) (2 mg/kg), an adenosine A(1)/A(2) receptor antagonist, attenuated hypotension induced by uridine and blocked the arterial pressure decrease in response to cytidine. Uridine-induced bradycardia was blocked by DMPX. 4-(2-[7-amino-2-(2-furyl[1,2,4]-triazolo[2,3-a[1,3,5]triazin-5-yl-aminoethyl)phenol (ZM241385) (1 mg/kg; i.v.), a selective adenosine A(2A) receptor antagonist, pretreatment produced an only very small blockade in the first minute of the hypotensive effects of uridine without affecting the bradycardia. ZM241385 pretreatment completely blocked cytidine's hypotensive effect. In Langendorff-perfused rat heart preparation, uridine (10(-3) M), but not cytidine, decreased the heart rate. Our results show that intravenously injected uridine or cytidine is able to decrease arterial pressure by activating peripheral adenosine receptors. The data also implicates that the mainly adenosine A(1) receptor activation is involved in the uridine-induced cardiovascular effects, while both adenosine A(1) and A(2A) receptor activations mediate the cytidine's effects.

Protective effect of adenosine in diabetic neuropathic pain is mediated through adenosine A1-receptors.

Diabetic neuropathic pain is generally considered to be one of the most troublesome complications affecting diabetic patients and current therapy provides inadequate pain relief. In the present study, the effect of adenosine was investigated in a model of diabetic neuropathic pain. Diabetes was induced by streptozotocin (65 mg/kg, ip) in male Sprague Dawley rats and subjected to thermal (cold and hot) and chemical (formalin) stimuli. Diabetic rats developed hyperalgesia by the end of six weeks in thermal and chemical stimuli test. Adenosine (100, 200 and 500 mg/kg, ip) produced significant reversal of responses to thermal and chemical stimuli in diabetic rats. 8-Cyclopentyl-1, 3-dipropylxanthine (DPCPX 1 mg/kg, ip), an adenosine A1-receptor antagonist, but not 3,7-dimethyl-1-propargylxanthine (DMPX 1 mg/kg, ip), an adenosine A2A-receptor antagonist, reversed the protective effect of adenosine. These results indicate that adenosine is an effective analgesics in a model of diabetic neuropathy, and the protection produced by adenosine is via stimulation of adenosine A1-receptors.

Morphometric and functional abnormalities of kidneys in the progeny of mice fed chocolate during pregnancy and lactation.

Even most commonly consumed beverages like tea, coffee, chocolate and cocoa contain methylxanthines, biogenic amines and polyphenols, among them catechins, that exhibit significant biological activity and might profoundly affect the organism homeostasis. We have previously shown that 400 mg of bitter chocolate or 6 mg of theobromine added to the daily diet of pregnant and afterwards lactating mice affected embryonic angiogenesis and caused bone mineralization disturbances as well as limb shortening in 4-weeks old offspring. The aim of the present study was the morphometric and functional evaluation of kidneys in the 4-weeks old progeny mice fed according to the protocol mentioned above. Progeny from the mice fed chocolate presented considerable morphometric abnormalities in the kidney structure, with the lower number of glomeruli per mm2 and their increased diameter. Moreover, higher serum creatinine concentration was observed in that group of offspring. No morphometric or functional irregularities were found in the progeny of mice fed theobromine. Abnormalities demonstrated in the offspring of mice fed chocolate are not related to its theobromine content. Consequently, identification of active compound(s) responsible for the observed effects is of vital importance.

Intrahepatic adenosine-mediated activation of hepatorenal reflex is via A1 receptors in rats.

Previous studies have shown that intrahepatic adenosine is involved in activation of the hepatorenal reflex that regulates renal sodium and water excretion. The present study aims to determine which subtype of adenosine receptors is implicated in the process. Mean arterial pressure, portal venous pressure and flow, and renal arterial flow were monitored in pentobarbital anesthetized rats. Urine was collected from the bladder. Intraportal administration of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist, increased urine flow by 24%, 89%, and 143% at the dose of 0.01, 0.03, and 0.1 mg x kg(-1), respectively; in contrast, DPCPX, when administered intravenously at the same doses, only increased urine flow by 0%, 18%, and 36%. The increases in urine flow induced by intraportal administration of DPCPX were abolished in rats with liver denervation. Intrahepatic infusion of adenosine significantly decreased urine flow and this response was abolished by intraportal administration of DPCPX. Neither intraportal nor intravenous administration of 3,7-dimethyl-1-propargylxanthine, a selective adenosine A2 receptor antagonist, showed significant influence on urine flow. Systemic arterial pressure, renal blood flow and glomerular filtration rate were unaltered by the administration of any of the drugs. In conclusion, intrahepatic adenosine A1 receptors are responsible for the adenosine-mediated hepatorenal reflex that regulates renal water and sodium excretion.

Multiple effects of theobromine on fetus development and postnatal status of the immune system.

Caffeine and its active derivative, theobromine, are probably the most frequently ingested pharmacologically active substances. Considering their uninhibited transport via the placental barrier as well as immature enzymatic activities and metabolic pathways in embryos and infants resulting in the longer half-life of methyloxanthines and their accumulation, unrestrained uptake of these substances might result in noticeably more pronounced biological effects during pregnancy and the postnatal period. Our previous studies have shown that methyloxanthines are significant inhibitors of angiogenic growth factors production and angiogenesis itself. We have hypothesized that increased uptake of these substances might affect embryonal angiogenesis and, later in the postnatal period, maturation and functional activity of the offspring's immune system. The study was performed on 2-month-old Balb/c mice fed theobromine 2 or 6 mg/day during pregnancy and lactation. On day 18 of pregnancy the number and weight of embryos were assessed as was their tissue angiogenic activity, using the cutaneous angiogenesis assay. In the group of 4-week-old sucklings, body and spleen were weighed together with the trunk, and tail and limb length were measured. Six weeks after birth the splenocytes' mitogen-induced activity and their ability to induce graft-versus-host reaction as well as the humoral response to SRBC antigen were evaluated. Content of theobromine in the embryos' tissue was estimated by high liquid performance chromatography (HPLC). Theobromine feeding resulted in significant inhibition of embryo growth as assessed by their weight and decreased angiogenic activity of their tissue. The theobromine content in embryo tissue from treated groups was higher than in the controls, and the difference was close to significant. In the postnatal period the discrepancies in the treated 4-week-old group's development were also observed in the significantly shorter limbs in comparison to the controls. Moreover in the treated group of 6-week-old sucklings, considerable variations in the immune system's functional activity were registered as far as cellular and immune response were concerned. Respectively, the splenocytes' mitogen-induced proliferative activity was significantly suppressed while the graft-versus-host reaction was up-regulated, and the serum antibodies titer was elevated in correspondence to the observed spleen enlargement. We concluded that a theobromine-enriched diet affects progeny development in both prenatal and postnatal periods. Consequently, particular attention should be paid to the reduction of theobromine consumption, and most probably that of other methyloxanthines, during pregnancy and lactation.

Roles of adenosine receptors in the regulation of kainic acid-induced neurotoxic responses in mice.

Kainic acid (KA) is a well-known excitatory and neurotoxic substance. In ICR mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the hippocampal CA3 pyramidal neurons. In the present study, the possible role of adenosine receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. It has been shown that 3,7-dimethyl-1-propargylxanthine (DMPX; A2 adenosine receptors antagonist, 20 microg) reduced KA-induced CA3 pyramidal cell death. KA dramatically increased the phosphorylated extracellular signal-regulated kinase (p-ERK) immunoreactivities (IR) in dentate gyrus (DG) and mossy fibers. In addition, c-Jun, c-Fos, Fos-related antigen 1 (Fra-1) and Fos-related antigen 2 (Fra-2) protein levels were increased in hippocampal area in KA-injected mice. DMPX attenuated KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. However, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX; A1 adenosine receptor antagonist, 20 microg) did not affect KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. KA also increased the complement receptor type 3 (OX-42) IR in CA3 region of hippocampus. DMPX, but not PACPX, blocked KA-induced OX-42 IR. Our results suggest that p-ERK and c-Jun may function as important regulators responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Activated microglia, which was detected by OX-42 IR, may be related to phagocytosis of degenerated neuronal elements by KA excitotoxicity. Furthermore, it is implicated that A2, but not A1, adenosine receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

Uncertainty factors for chemical risk assessment: interspecies differences in the in vivo pharmacokinetics and metabolism of human CYP1A2 substrates.

The 100-fold default uncertainty factor is used to convert a no-observed-adverse-effect level (NOAEL) from a animal toxicity study, to a "safe" value for human intake. The composite uncertainty factor (100) has to allow for interspecies (10-fold) and interindividual (10-fold) differences in toxicokinetics and toxicodynamics. The aim of the current study was to assess the validity of the interspecies default for toxicokinetics (4.0) for each of the test species (dog, rabbit, rat and mouse), using published data for compounds eliminated by CYP1A2 in humans (caffeine, theobromine, theophylline and paraxanthine). An analysis of the published literature showed that the absorption, bioavailability and route of excretion were generally similar between humans and the test species, for each probe substrate. However, interspecies differences in the route of metabolism, and the enzymes involved in this process, were identified. The magnitude of difference in the internal dose, between species, showed that values for the mouse (10.6) and rat (5.4) exceed the 4.0-fold default, whereas the rabbit (2.6) and dog (1.6) were below this value. This work supports the need to replace the generic default factors by a compound-related value derived from specific, relevant, quantitative data; this would result in more relevant and reliable non-cancer risk assessments.

Neuroprotection by caffeine and A(2A) adenosine receptor inactivation in a model of Parkinson's disease.

Recent epidemiological studies have established an association between the common consumption of coffee or other caffeinated beverages and a reduced risk of developing Parkinson's disease (PD). To explore the possibility that caffeine helps prevent the dopaminergic deficits characteristic of PD, we investigated the effects of caffeine and the adenosine receptor subtypes through which it may act in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model of PD. Caffeine, at doses comparable to those of typical human exposure, attenuated MPTP-induced loss of striatal dopamine and dopamine transporter binding sites. The effects of caffeine were mimicked by several A(2A) antagonists (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261), 3,7-dimethyl-1-propargylxanthine, and (E)-1,3-diethyl-8 (KW-6002)-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione) (KW-6002) and by genetic inactivation of the A(2A) receptor, but not by A(1) receptor blockade with 8-cyclopentyl-1,3-dipropylxanthine, suggesting that caffeine attenuates MPTP toxicity by A(2A) receptor blockade. These data establish a potential neural basis for the inverse association of caffeine with the development of PD, and they enhance the potential of A(2A) antagonists as a novel treatment for this neurodegenerative disease.